The long-term goal of the research described in this application is to find ways to improve vaccines against epidemic influenza in the human population. The objective of the proposed experiments is to gain better understanding of how antibodies neutralize virus infectivity and the mechanisms by which influenza A viruses escape from neutralizing antibodies. We will focus effort on the viral neuraminidase (NA) since this is the only viral antigen for which a crystal structure of an antigen-antibody complex is available. Also antibody inhibition of NA enzyme activity can be quantitatively measured. However, we have not forgotten that the hemagglutinin (HA) is more plentiful on the virus surface, has better-defined and multiple functions, and that anti-HA antibodies directly neutralize infectivity. Therefore we propose experiments to use the procedures we will have worked out for the NA to investigate mechanisms of neutralization by anti-HA antibodies. The Specific Aims are to answer the following questions: 1. How does the influenza neuraminidase escape from antibody inhibition? Site-directed mutagenesis procedures will be used to test the hypothesis that only some of the 22 amino acids on subtype N9 NA which make contact with antibody NC41 are essential to the interaction, and therefore that only certain changes can lead to antigenic drift. 2. Are the principles of virus escape deduced from N9 NA relevant to human influenza? The antigenic structure and variation of N2 NA from a recent human isolate, A/Shanghai/11/87 will be studied using monoclonal antibodies, following procedures developed for the avian virus N9 NA which have been validated by x-ray crystallographic results. 3. What is the mechanism of inhibition by anti-NA antibodies such as NC41? What function of influenza virus NA are they inhibiting? The x-ray crystal structure of NA-antibody complex suggests that inhibition is not necessarily stearic. The effect of antibody on the NA active site will be determined, and the role of NA as a receptor-destroying enzyme will be investigated. 4. Mechanism of neutralization by anti-HA antibodies. Antibodies against the HA are classical neutralizing antibodies, unlike those to the NA which act after infection has begun. We will apply the procedures developed for studying antibody inhibition of NA to localize neutralizing epitopes on a recent human HA.